Oligosaccharide analytical standards

ABSTRACT

Disclosed herein are oligosaccharides and intermediates useful for the production thereof. The compounds are useful as analytical standards and as intermediates for the preparation of more complex oligosaccharide and N-glycan products. The compounds may be prepared in high purity using the selective stop/go synthetic methods disclosed herein.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application62/945,613, filed Dec. 9, 2019, the contents of which are herebyincorporated in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under P01GM107012,P41GM103390, U01GM120408 and F31CA180478 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The invention is directed to synthesis of complex N-linkedoligosaccharide including methods of preparing key intermediates thatcan be readily elaborated into more complicated compounds such asglycoconjugates, and derivatives thereof for use as native orisotopically enriched analytical standards with application in massspectrometry, high-pressure liquid chromatography, capillaryelectrophoresis, and nuclear magnetic resonance; for antibody,glycoprotein, and therapeutic development; and for construction ofanalytical or high-throughput platforms including microarray andbiofluid analysis.

BACKGROUND

N-glycosylation of proteins is one of the most complex and diversepost-translational modifications that can influence a multitude ofbiological processes such as signal transduction, embryogenesis,neuronal development, fertilization, hormone activity, immune regulationand the proliferation of cells and their organization into specifictissues. It has been implicated in the etiology of many human diseasessuch as pathogen recognition, inflammation, immune responses, thedevelopment of autoimmune diseases, and cancer. Although it is widelyaccepted that N-glycans contain high information content, the limitedaccessibility of well-defined structures makes it difficult to uncoverthe molecular basis by which they regulate biological and diseaseprocesses. Consequently, diverse collections of well-defined N-glycansare needed as standards for glycan structure determination ofheterogeneous biological samples, as ligands to study interactions withglycan-binding proteins, as probes to examine the molecular basis ofglycoconjugate biosynthesis and as starting materials for glycoproteinsynthesis.

There remains a need for improved methods of synthesizingoligosaccharides, including those found in biologically relevantsystems, including glycans. There remains a need for analyticalstandards permitting the rapid identification and quantification ofN-glycans in a biomolecule of interest.

FIG. 1 depicts Structure of N-glycans and a bio-inspired strategy fortheir preparation. FIG. 1a , MGAT enzymes responsible for installingGlcNAc at different branching points. FIG. 1b , Enzyme classes involvedin the biosynthesis of complex N-glycans. FIG. 1c , Structure ofunnatural UDP-GlcNTFA (4). FIG. 1d , Bio-inspired strategy for thesynthesis of asymmetric N-glycans. Symmetrical bi-antennary glycan 1,which can easily be obtained from a glycopeptide isolated from egg yolk,can be further branched by recombinant MGAT4 and MGAT5. The use ofunnatural UDP-GlcNTFA makes it possible to prepare 2 bearing GlcNAc,GlcN₃ and GlcNH₂ branching moieties. Compound 2 is the key intermediatefor preparing complex targets such as 3. FIG. 1e , Transformation ofGlcNTFA, installed by MGAT4 and MGAT5, into GlcNH₂ or GlcN₃ ‘stops’further enzymatic extension of these moieties until they are convertedinto natural GlcNAc (‘go’), which can then be elaborated byglycosyltransferases into complex appendages.

FIG. 2 depicts the synthesis of asymmetric branched tri-antennaryglycosyl asparagines using MGAt5 and uDP-GlcNtFA. MGAT5 readily acceptsUDP-GlcNTFA to give a tri-antennary glycan that, following basetreatment, provides a compound with a GlcNH₂ at the β 6 arm. The latterresidue is not a substrate for the galactosyl transferase B4GalT1 andtherefore it is possible to selectively elaborate the MGAT1 and MGAT2arms by exploiting the inherent branch selectivities of glycosidases andglycosyltransferases. Once the MGAT1 and MGAT2 arms were capped withNeu5Ac, preventing these positions from further elongation, the GlcNH₂could be acetylated to give natural GlcNAc capable of being extended bya series of glycosyltransferases.

FIG. 3 depicts the synthesis of asymmetric branched tetra-antennaryN-glycans using MGAt4 and MGAt5 in combination with uDP-GlcNtFA andsubsequent conversion of the transferred GlcNtFA into GlcN₃ orGlcNH_(2.) The latter moieties are temporarily disabled frommodification by glycosyltransferases, making it possible to selectivelyelaborate the MGAT1 and MGAT2 arms. At an appropriate point in thesynthesis, the unnatural GlcN₃ or GlcNH₂ moieties can be converted intonatural GlcNAc, allowing each arm to be uniquely extended.

FIG. 4 depicts additional compounds of the invention. The symbols usedto depict sugars is presented in FIG. 1.

FIG. 5 depicts additional compounds of the invention. The symbols usedto depict sugars is presented in FIG. 1.

FIG. 6 depicts additional compounds of the invention. The symbols usedto depict sugars is presented in FIG. 1.

DETAILED DESCRIPTION

Before the present methods and systems are disclosed and described, itis to be understood that the methods and systems are not limited tospecific synthetic methods, specific components, or to particularcompositions. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Ranges may be expressed herein as from “about” oneparticular value, and/or to “about” another particular value. When sucha range is expressed, another embodiment includes¬from the oneparticular value and/or to the other particular value. Similarly, whenvalues are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms anotherembodiment. It will be further understood that the endpoints of each ofthe ranges are significant both in relation to the other endpoint, andindependently of the other endpoint.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers or steps.

“Exemplary” means “an example of” and is not intended to convey anindication of a preferred or ideal embodiment. “Such as” is not used ina restrictive sense, but for explanatory purposes.

Disclosed are components that can be used to perform the disclosedmethods and systems. These and other components are disclosed herein,and it is understood that when combinations, subsets, interactions,groups, etc. of these components are disclosed that while specificreference of each various individual and collective combinations andpermutation of these may not be explicitly disclosed, each isspecifically contemplated and described herein, for all methods andsystems. This applies to all aspects of this application including, butnot limited to, steps in disclosed methods. Thus, if there are a varietyof additional steps that can be performed it is understood that each ofthese additional steps can be performed with any specific embodiment orcombination of embodiments of the disclosed methods.

The term “alkyl” as used herein is a branched or unbranched hydrocarbongroup such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, and thelike. The alkyl group can also be substituted or unsubstituted. Unlessstated otherwise, the term “alkyl” contemplates both substituted andunsubstituted alkyl groups. The alkyl group can be substituted with oneor more groups including, but not limited to, alkoxy, alkenyl, alkynyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aldehyde, amino,carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl,sulfo-oxo, or thiol. An alkyl group which contains no double or triplecarbon-carbon bonds is designated a saturated alkyl group, whereas analkyl group having one or more such bonds is designated an unsaturatedalkyl group. Unsaturated alkyl groups having a double bond can bedesignated alkenyl groups, and unsaturated alkyl groups having a triplebond can be designated alkynyl groups. Unless specified to the contrary,the term alkyl embraces both saturated and unsaturated groups.

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, etc. The term “heterocycloalkyl” is a cycloalkyl group asdefined above where at least one of the carbon atoms of the ring isreplaced with a heteroatom such as, but not limited to, nitrogen,oxygen, sulfur, selenium or phosphorus. The cycloalkyl group andheterocycloalkyl group can be substituted or unsubstituted. Unlessstated otherwise, the terms “cycloalkyl” and “heterocycloalkyl”contemplate both substituted and unsubstituted cyloalkyl andheterocycloalkyl groups. The cycloalkyl group and heterocycloalkyl groupcan be substituted with one or more groups including, but not limitedto, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,hydroxy, ketone, nitro, silyl, sulfo-oxo, or thiol. A cycloalkyl groupwhich contains no double or triple carbon-carbon bonds is designated asaturated cycloalkyl group, whereas a cycloalkyl group having one ormore such bonds (yet is still not aromatic) is designated an unsaturatedcycloalkyl group. Unless specified to the contrary, the term cycloalkylembraces both saturated and unsaturated, non-aromatic, ring systems.

The term “aryl” as used herein is an aromatic ring composed of carbonatoms. Examples of aryl groups include, but are not limited to, phenyland naphthyl, etc. The term “heteroaryl” is an aryl group as definedabove where at least one of the carbon atoms of the ring is replacedwith a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur,selenium or phosphorus. The aryl group and heteroaryl group can besubstituted or unsubstituted. Unless stated otherwise, the terms “aryl”and “heteroaryl” contemplate both substituted and unsubstituted aryl andheteroaryl groups. The aryl group and heteroaryl group can besubstituted with one or more groups including, but not limited to,alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,hydroxy, ketone, nitro, silyl, sulfo-oxo, or thiol.

Exemplary heteroaryl and heterocyclyl rings include: benzimidazolyl,benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl,benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aHcarbazolyl, carbolinyl, chromanyl, chromenyL cirrnolinyl,decahydroquinolinyl, 2H,6H˜1,5,2-dithiazinyl, dihydrofuro[2,3b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl,imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl,3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl,isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,methylenedioxyphenyl, morpholinyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl.

The terms “alkoxy,” “cycloalkoxy,” “heterocycloalkoxy,” “cycloalkoxy,”“aryloxy,” and “heteroaryloxy” have the aforementioned meanings foralkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl, furtherproviding said group is connected via an oxygen atom.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, and aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described below. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this disclosure, the heteroatoms, such as nitrogen, canhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valencies of theheteroatoms. This disclosure is not intended to be limited in any mannerby the permissible substituents of organic compounds. Also, the terms“substitution” or “substituted with” include the implicit proviso thatsuch substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., a compound that does not spontaneouslyundergo transformation such as by rearrangement, cyclization,elimination, etc. Unless specifically stated, a substituent that is saidto be “substituted” is meant that the substituent can be substitutedwith one or more of the following: alkyl, alkoxy, alkenyl, alkynyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aldehyde, amino,carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl,sulfo-oxo, or thiol. In a specific example, groups that are said to besubstituted are substituted with a protic group, which is a group thatcan be protonated or deprotonated, depending on the pH.

The skilled person will understand that when the disclosed compoundsbear ionizable functional groups (e.g., amines, carboxylic acids,sulfonic acids, etc) the compounds may be protonated or deprotonateddepending on pH. Unless specifically stated otherwise, the structure ofa compound embraces all ionized forms of the compound as well.Acceptable salts of the disclosed compounds may be formed underconventional conditions. Examples of such salts are acid addition saltsformed with inorganic acids, for example, hydrochloric, hydrobromic,sulfuric, phosphoric, and nitric acids and the like; salts formed withorganic acids such as acetic, oxalic, tartaric, succinic, maleic,fumaric, gluconic, citric, malic, methanesulfonic, p-toluenesulfonic,napthalenesulfonic, and polygalacturonic acids, and the like; saltsformed from elemental anions such as chloride, bromide, and iodide;salts formed from metal hydroxides, for example, sodium hydroxide,potassium hydroxide, calcium hydroxide, lithium hydroxide, and magnesiumhydroxide; salts formed from metal carbonates, for example, sodiumcarbonate, potassium carbonate, calcium carbonate, and magnesiumcarbonate; salts formed from metal bicarbonates, for example, sodiumbicarbonate and potassium bicarbonate; salts formed from metal sulfates,for example, sodium sulfate and potassium sulfate; and salts formed frommetal nitrates, for example, sodium nitrate and potassium nitrate.

Disclosed herein are a family of oligosaccharides and intermediatesuseful as analytical standards, and for applications such asglycopeptide synthesis, microarray development, and others. Theoligosaccharides can have the formula:

-   -   wherein R¹ and R⁴ are as defined herein, R^(MG2), R^(MG3),        R^(MG4), and R^(MG5), are each independently chosen from H,        GlcNAc, or modified GlcNAc. As used herein, a modified GlcNAc        refers to a 2-deoxygluco residue bearing substituted or        unsubstituted nitrogen atom at the 2-position.

In some embodiments, the invention relates to a modified GlcNAc glycosyldonor having the formula:

-   -   wherein PN represents a phosphonucleotide and PG represents a        protective group that is tolerated by glycosyltransferase        enzymes. After glycosylation, the protective group can be        removed to yield the corresponding 2-deoxyglucosamine, which can        be further derivatized using appropriate chemistries. Because        the 2-deoxyglucosamine and derivatives thereof are not        substrates for galactosyltransferase, the present invention        provides methods of selectively preparing a vast number of        different oligosaccharide compounds.

Disclosed herein are oligosaccharide compounds having the formula:

-   -   and salts thereof, wherein    -   R¹ can be OH, or a residue having the formula:

-   -   wherein R^(oz) can be H, C₁₋₄alkyl (preferably CH₃), aryl, CF₃,        CCl₃,    -   n can be 1 or 0;    -   R^(fa) can be H or a fucose residue having the structure:

-   -   R² can be OR^(c), NR^(2n)OR^(c); NR^(2n)R^(c) and R³ can be        OR^(c) or R^(c);        -   wherein:        -   R^(2n) can be H and C₁₋₄alkyl;        -   R^(c) can be X^(p)H, X^(p)C₁₋₈alkyl, X^(p)C₁₋₈alkylaryl,            X^(p)aryl, X^(p)fluorescent marker, or an amino acid residue            having the formula:

-   -   wherein R^(aa1) and R^(aa2) can be H or additional amino acid        residues, for instance as found in a protein, polypeptide,        monoclonal antibody, etc. In certain embodiments, R^(aa1) can be        Cbz (carboxybenzyl ether), and R^(aa2) can be H.    -   R³ can be X^(p)C₁₋₈alkyl, X^(p)C₁₋₈alkylaryl, or X^(p)aryl;    -   wherein X^(p) can be null or a polymer, preferably a        polyethylene glycol —(CH₂CH₂O)_(z)—, wherein z is 1-500.    -   R⁴ can be N₃, NR^(4a)R^(4b),    -   wherein:    -   R^(4a) and R^(4b) are the same or different, and can be H, Z—X⁴        wherein Z can be null, C═O, SO₂, and    -   X⁴ can be C₁₋₄alkyl, O—C₁₋₄alkyl, C₁₋₄haloalkyl,        O—C₁₋₄haloalkyl, C₁₋₄alkylaryl, O—C₁₋₄alkylaryl,    -   aryl, O-aryl; or R^(4a) and R^(4b) can together form a ring; or        R⁴ can be a radical having the formula:

-   -   and one or both of R^(4c) or R^(4d) constitute a conjugated        payload.

In certain embodiments, R^(4a) can be H and R^(4b) can be methoxymethyl,methylthiomethyl, p-methoxybenzyloxymethyl, p-nitrobenzyloxymethyl,t-butoxymethyl, 2-methoxyethoxymethyl, 1-ethoxyethyl, allyl,p-methoxybenzyloxycarbonyl (Moz), p-nitrobenzyloxycarbonyl (PNZ),trimethylsilyl, diethylisopropylsilyl, triphenylsilyl, formyl,chloroacetyl, methanesulfonyl, tosyl, benzylsulfonyl,methoxymethylcarbonyl, benzyloxycarbonyl, carboxybenzyl (Cbz),t-butyloxycarbonyl (BOC), 9-fluorenylmethylcarbonyl, N-phenylcarbamoyl,[2-(trimethylsilyl)ethoxy]methyl, or 4,4′-dimethoxytrityl.

While R², when present, can be in either the α or β configuration, or asa mixture of anomers, it is be preferred that R² is in the βconfiguration:

In some instances, n can be zero, e.g., R¹ is a residue having theformula:

In other embodiments, n is one, e.g., R¹ is a residue having theformula:

In some instances, R^(c) can be C₁₋₈alkyl, C₁₋₈alkylaryl, a polymer suchas polyethylene glycol, or aryl bearing functional group enablingcovalent or affinity-based immobilization for a microarray slide. Forinstance, R^(c) can have the formula —(CH₂)_(nc)R^(im), wherein nc is aninteger from 1-8, and R^(im) can be —NH₂, —SH, —OH, —COOH, —N₃, C≡CH, aMichael acceptor such as vinyl sulfone or maleimide, SO₃, OSO₃, or abiotin residue having the formula:

-   -   wherein X^(bt) is selected from null, O, NH, or S.

In other instances, R^(c) can be a group suitable for UV-VIS orfluorescent detection. Suitable aryl groups typically includepolyaromatic systems such as coumarins, rhodamines, fluoresceins,cyanines, eosins, erythrosins, and the like.

In other instances, R^(c) can be a C₁₋₈alkylaryl or aryl group suitablefor solid phase extractions. Exemplary aryl groups include phenyls andnaphthyls bearing two or more sulfonate residues:

-   -   wherein np is from 0-50, ns is from 2-7, and ns′ is from 2-5,        with two sulfonate groups in an ortho configuration being        particularly preferred:

In some embodiments, R^(c) can be an aryl group useful as a tag isLC-MS, microarray, or capillary electrophoresis analysis. For exampleR^(c) can be an aryl (e.g., phenyl, naphthyl, anthracene, phenanthrene,phenalene, tetracene, chrysene, triphenylene, pyrene and the like)residue substituted one or more times by carboxylic acids, carboxamides(especially primary carboxamides), sulfonates, reporter groups such asquaternary ammonium salts and the like. By way of example, suitable tagsinclude residues having the formula:

-   -   wherein ne is from 2-8.

Also disclosed herein are oligosaccharides having the formula:

-   -   wherein R¹ and R⁴ are as defined above, and R⁵ can be N₃,        NR^(5a)R^(5b), wherein R^(5a) and R^(5b) are the same or        different, and can be H, Z—X⁵ wherein Z can be null, C═O, SO₂,        and X⁴ can be C₁₋₄alkyl, O—C₁₋₄alkyl, C₁₋₄haloalkyl,        O—C₁₋₄haloalkyl, C₁₋₄alkylaryl, O—C₁₋₄alkylaryl, aryl, O-aryl;        or R^(5a) and R^(5b) can together form a ring; or R⁵ can be a        radical having the formula:

-   -   wherein one or both of R^(5c) or R^(5d) constitute a conjugated        payload.

In certain embodiments, R^(5a) can be H and R^(5b) can be methoxymethyl,methylthiomethyl, p-methoxybenzyloxymethyl, p-nitrobenzyloxymethyl,t-butoxymethyl, 2-methoxyethoxymethyl, 1-ethoxyethyl, allyl,p-methoxybenzyloxycarbonyl (Moz), p-nitrobenzyloxycarbonyl (PNZ),trimethylsilyl, diethylisopropylsilyl, triphenylsilyl, formyl,chloroacetyl, methanesulfonyl, tosyl, benzylsulfonyl,methoxymethylcarbonyl, benzyloxycarbonyl, carboxybenzyl (Cbz),t-butyloxycarbonyl (BOC), 9-fluorenylmethylcarbonyl, N-phenylcarbamoyl,[2-(trimethylsilyl)ethoxy]methyl, or 4,4′-dimethoxytrityl.

In certain embodiments, the R⁵ bearing residue can be an isotopicallyenriched GlcNAc or modified GlcNAc, for instance in which one or more ofthe carbon atoms are enriched with ¹³C above naturally occurring levels.In some embodiments, R⁵ bearing residue is a ¹³C₆ enriched residue,which herein means that each of the ring carbons and the C-6 carbon areenriched with ¹³C above naturally occurring levels. When R⁵ is NHCOCH₃,one or both of those carbon atoms may also be ¹³C enriched. Suchresidues are termed ¹³C₇ and ¹³C₈ enriched residues, respectively. Theisotopic enrichment can be at least 90%, at least 95%, at least 98%, orat least 99%.

Also disclosed herein are oligosaccharides having the formula:

-   -   wherein R¹, R⁴, and R⁵ are as defined above;    -   R⁶ can be hydrogen or a residue having the formula:

-   -   wherein R⁹ can be N₃, NR^(9a)R^(9b), wherein R^(9a) and R^(9b)        are the same or different, and can be H, Z—X⁹ wherein Z can be        null, C═O, SO₂, and X⁹ can be C₁₋₄alkyl, O—C₁₋₄alkyl,        C₁₋₄haloalkyl, O—C₁₋₄haloalkyl, C₁₋₄alkylaryl, O—C₁₋₄alkylaryl,        aryl, O-aryl; or R^(9a) and R^(9b) can together form a ring; or    -   R⁹ can be a radical having the formula:

-   -   wherein one or both of R^(9c) or R^(9d) constitute a conjugated        payload.

In certain embodiments, R^(9a) can be H and R^(9b) can be methoxymethyl,methylthiomethyl, p-methoxybenzyloxymethyl, p-nitrobenzyloxymethyl,t-butoxymethyl, 2-methoxyethoxymethyl, 1-ethoxyethyl, allyl,p-methoxybenzyloxycarbonyl (Moz), p-nitrobenzyloxycarbonyl (PNZ),trimethylsilyl, diethylisopropylsilyl, triphenylsilyl, formyl,chloroacetyl, methanesulfonyl, tosyl, benzylsulfonyl,methoxymethylcarbonyl, benzyloxycarbonyl, carboxybenzyl (Cbz),t-butyloxycarbonyl (BOC), 9-fluorenylmethylcarbonyl, N-phenylcarbamoyl,[2-(trimethylsilyl)ethoxy]methyl, or 4,4′-dimethoxytrityl. R⁷ can behydrogen or a residue having the formula:

-   -   wherein R¹⁰ can be N₃, NR^(10a)R^(10b), wherein R^(10a) and        R^(10b) are the same or different, and can be H, Z—X¹⁰ wherein Z        can be null, C═O, SO₂, and X⁴ can be C₁₋₄alkyl, O—C₁₋₄alkyl,        C₁₋₄haloalkyl, O—C₁₋₄haloalkyl, C₁₋₄alkylaryl, O—C₁₋₄alkylaryl,        aryl, O-aryl; or R^(10a) and R^(10b) can together form a ring;        or R¹⁰ can be a radical having the formula:

-   -   wherein one or both of R^(10c) or R^(10d) constitute a        conjugated payload.

In certain embodiments, R^(10a) can be H and R^(10b) can bemethoxymethyl, methylthiomethyl, p-methoxybenzyloxymethyl,p-nitrobenzyloxymethyl, t-butoxymethyl, 2-methoxyethoxymethyl,1-ethoxyethyl, allyl, p-methoxybenzyloxycarbonyl (Moz),p-nitrobenzyloxycarbonyl (PNZ), trimethylsilyl, diethylisopropylsilyl,triphenylsilyl, formyl, chloroacetyl, methanesulfonyl, tosyl,benzylsulfonyl, methoxymethylcarbonyl, benzyloxycarbonyl, carboxybenzyl(Cbz), t-butyloxycarbonyl (BOC), 9-fluorenylmethylcarbonyl,N-phenylcarbamoyl, [2-(trimethylsilyl)ethoxy]methyl, or4,4′-dimethoxytrityl.

R¹² can be hydrogen or a residue having the formula:

-   -   wherein R¹³ can be hydrogen or a residue having the formula:

-   -   wherein R^(13b) is selected from H and a conjugated cargo        moiety, and R^(13a) is selected from OH and a conjugated cargo        moiety;    -   R¹⁴ can be hydrogen or a residue having the formula:

-   -   wherein R^(14b) is selected from H and a conjugated cargo        moiety, and R^(14a) is selected from OH and a conjugated cargo        moiety;    -   R⁸ can be hydrogen or a residue having the formula:

-   -   wherein R¹¹ can be N₃, NR^(11a)R^(11b) wherein R^(11a) and        R^(11b) are the same or different, and can be H, Z—X¹¹ wherein Z        can be null, C═O, SO₂, and X¹¹ can be C₁₋₄alkyl, O—C₁₋₄alkyl,        C₁₋₄haloalkyl, O—C₁₋₄haloalkyl, C₁₋₄alkylaryl, O—C₁₋₄alkylaryl,        aryl, O-aryl; or R^(11a) and R^(11b) can together form a ring;        or R¹¹ can be a radical having the formula:

-   -   wherein one or both of R^(11c) or R^(11d) constitute a        conjugated payload.

In certain embodiments, R^(11a) can be H and R^(11b) can bemethoxymethyl, methylthiomethyl, p-methoxybenzyloxymethyl,p-nitrobenzyloxymethyl, t-butoxymethyl, 2-methoxyethoxymethyl,1-ethoxyethyl, allyl, p-methoxybenzyloxycarbonyl (Moz),p-nitrobenzyloxycarbonyl (PNZ), trimethylsilyl, diethylisopropylsilyl,triphenylsilyl, formyl, chloroacetyl, methanesulfonyl, tosyl,benzylsulfonyl, methoxymethylcarbonyl, benzyloxycarbonyl, carboxybenzyl(Cbz), t-butyloxycarbonyl (BOC), 9-fluorenylmethylcarbonyl,N-phenylcarbamoyl, [2-(trimethylsilyl)ethoxy]methyl, or4,4′-dimethoxytrityl.

Disclosed herein are oligosaccharides having the formula:

-   -   wherein R¹, R⁴, R⁵, R⁶, R⁷, and R⁸ have the meanings given        above;    -   R¹⁵ can hydrogen or a residue having the formula:

-   -   wherein R¹⁷ can be hydrogen or a residue having the formula:

-   -   wherein R^(17b) can be H or a conjugated cargo moiety, and        R^(17a) can be OH or a conjugated cargo moiety;    -   R¹⁸ can be hydrogen or a residue having the formula:

-   -   wherein R^(18b) is can be H or a conjugated cargo moiety, and        R^(18a) can be OH or a conjugated cargo moiety;    -   R¹⁶ can be hydrogen or a residue having the formula:

-   -   wherein R¹⁹ can be hydrogen or a residue having the formula:

-   -   wherein R^(18b) can be H or a conjugated cargo moiety, and        R^(18a) can be OH or a conjugated cargo moiety;    -   R²⁰ can be hydrogen or a residue having the formula:

-   -   wherein R^(20b) can be H or a conjugated cargo moiety, and        R^(20a) can be OH and a conjugated cargo moiety.

As used herein, a conjugated payload in the context of the triazoleresidues described above can be formed using click chemistrycycloadditions between an azide and alkyl or alkene:

-   -   wherein Y is a cytotoxic drug or tracer compound, and L is a        linker. In some instances the conjugated payload/triazole can        have the formula:

-   -   wherein R⁰ is in each case independently selected from hydrogen,        halogen, C₁₋₈alkyl, C₁₋₈alkoxy, aryl, C₁₋₈heteroaryl,        C₃₋₈cycloalkyl, or C₁₋₈heterocyclyl; wherein any two or more R⁰        groups can together form a ring;    -   E¹ can be:

-   -   wherein L^(C1) can be null, cleavable linker, and non-cleavable        linker. In some embodiments, the conjugated payload/triazole can        have the formula:

-   -   wherein R^(e) is selected from hydrogen, or either *—OSO₃X¹ or        *—OPO₃X¹, wherein X¹ is selected from H, C₁₋₈alkyl, or a        pharmaceutically acceptable cation. In other embodiments, each        of R⁰ is hydrogen.

Compounds disclosed herein can be prepared from by selectivelyglycosylating an appropriate acceptor with a GlcNHC(O)R^(a) donor asshown below:

-   -   wherein R¹ has the meaning given above, and R^(a) is CX₃, CHX₂,        CH₂X, CH₃, OBn, wherein X is in each case independently selected        from F, Cl, Br, and I, and UDP is a residue having the formula:

The above glycosylation may be carried out using a suitableglycosyltransferase, for instance α-1,3-mannosyl-glycoprotein2-β-N-acetylglucosaminyltransferase, referred to herein as MGAT1.

In certain embodiments, when R^(a) is CX₃, CHX₂, CH₂X, or OBn, thehaloacetamide or Cbz group can be removed to furnish the free amine,which can be further elaborated to R⁴ as defined herein:

In some embodiments, R⁴ can be azide. After glycosylation with MGAT1,the resulting product (either with or without conversion to the freeamine or the R⁴ group) can be selectively glycosylated with a furtherGlcNHC(O)R^(a) donor:

The above glycosylation may be carried out using a suitableglycosyltransferase, for instance α-1,6-mannosyl-glycoprotein2-β-N-acetylglucosaminyltransferase, referred to herein as MGAT2. Inpreferred embodiments, R⁴ and GlcNHC(O)R^(a) are not the same. Theresulting oligosaccharide can be further elaborated as described above:

The disclosed compounds are useful intermediates for a variety ofadditional selective transformations: Use of any of MGAT3, MGAT4, and/orMGAT5 permits selective installation of GlcNHC(O)R^(a) residues on theoligosaccharide, which can be converted to R⁶, R⁷, and R⁸ residues asdefined above.

As R⁴ and R⁵ can be selected to deactivate those GlcNAc derivatives togalactosyltransferases (e.g., when neither R⁴ nor R⁵ are NHC(O)R^(a),selective elaboration of the oligosaccharide at other arms of the glycancan be achieved:

Also disclosed herein are oligosaccharides having the formula:

-   -   wherein R¹, R⁴, and R⁵ are as defined above;    -   R⁶ can be hydrogen or a residue having the formula:

-   -   wherein R⁹ can be N₃, NR^(9a)R^(9b), wherein R^(9a) and R^(9b)        are the same or different, and can be H, Z—X⁹ wherein Z can be        null, C═O, SO₂, and X⁹ can be C₁₋₄alkyl, O—C₁₋₄alkyl,        C₁₋₄haloalkyl, O—C₁₋₄haloalkyl, C₁₋₄alkylaryl, O—C₁₋₄alkylaryl,        aryl, O-aryl; or R^(9a) and R^(9b) can together form a ring; or    -   R⁹ can be a radical having the formula:

-   -   wherein one or both of R^(9c) or R^(9d) constitute a conjugated        payload.

In certain embodiments, R^(9a) can be H and R^(9b) can be methoxymethyl,methylthiomethyl, p-methoxybenzyloxymethyl, p-nitrobenzyloxymethyl,t-butoxymethyl, 2-methoxyethoxymethyl, 1-ethoxyethyl, allyl,p-methoxybenzyloxycarbonyl (Moz), p-nitrobenzyloxycarbonyl (PNZ),trimethylsilyl, diethylisopropylsilyl, triphenylsilyl, formyl,chloroacetyl, methanesulfonyl, tosyl, benzylsulfonyl,methoxymethylcarbonyl, benzyloxycarbonyl, carboxybenzyl (Cbz),t-butyloxycarbonyl (BOC), 9-fluorenylmethylcarbonyl, N-phenylcarbamoyl,[2-(trimethylsilyl)ethoxy]methyl, or 4,4′-dimethoxytrityl.

Disclosed herein are compounds listed in the following table:

The compounds may be characterized by a natural isotopic abundance forthe indicated GlcNAc residue, or that residue may be ¹³C-enriched asdefined herein.

Compound 1 R^(1a), R^(1b), R^(1c), R², R³, R⁴, R⁵ H Compound 2 R^(1b),R^(1c), R², R³, R⁴, R⁵ H R^(1a)

Compound 3 R^(1b), R^(1c), R², R³, R⁵ H R^(1a)

R⁴

Compound 4 R^(1a), R^(1b), R^(1c), R², R⁵ H R³

R⁴

Compound 5 R^(1b), R^(1c), R², R⁵ H R^(1a)

R³

R⁴

Compound 6 R^(1a), R^(1b), R^(1c), R², R⁵ H R³

R⁴

Compound 7 R^(1a), R^(1b), R^(1c), R², R⁵ H R³

R⁴

Compound 8 R^(1a), R^(1b), R^(1c), R², R⁵ H R³

R⁴

Compound 9 R^(1a), R^(1b), R^(1c), R², R⁵ H R³

R⁴

Compound 10 R^(1a), R^(1b), R^(1c), R², R⁵ H R³

R⁴

Compound 11 R^(1a), R^(1b), R^(1c), R², R⁵ H R³

R⁴

Compound 12 R^(1b), R^(1c), R², R⁵ H R^(1a)

R³

R⁴

Compound 13 R^(1b), R^(1c), R², R⁵ H R^(1a)

R³

R⁴

Compound 14 R^(1b), R^(1c), R⁵ H R^(1a)

R²

R³

R⁴

Compound 15 R^(1b), R^(1c), R⁵ H R^(1a)

R²

R³

R⁴

Compound 16 R^(1b), R^(1c), R⁵ H R^(1a)

R²

R³

R⁴

Compound 17 R^(1a), R^(1b), R^(1c), R² H R³

R⁴

R⁵

Compound 18 R^(1a), R^(1b), R^(1c), R² H R³

R⁴

R⁵

Compound 19 R^(1a), R^(1b), R^(1c), R² H R³

R⁴

R⁵

Compound 20 R^(1a), R^(1b), R^(1c), R² H R³

R⁴

R⁵

Compound 21 R^(1a), R^(1b), R^(1c), R² H R³

R⁴

R⁵

Compound 22 R^(1a), R^(1b), R^(1c), R² H R³

R⁴

R⁵

Compound 23 R^(1a), R^(1b), R^(1c), R² H R³

R⁴

R⁵

Compound 24 R^(1a), R^(1b), R^(1c), R² H R³

R⁴

R⁵

The disclosed compounds are useful as analytical standard for thecharacterization and quantification of N-glycans. In some embodimentsare provided kits containing at least one, isolated compound in a vial.Preferably, the compound will be present in the vial as a lyophilizedmixture, optionally in combination with one or more inert bulking orstabilizing agents. The purity of the compound in the vial can be atleast 90%, at least 95%, at least 98%, or at least 99%, as measured byHPLC. Some kits may contain multiple vials, each containing a singlecompound different from the rest. Exemplary kits may include at least 2compounds, at least 3 compounds, at least 5 compounds, at least 8compounds, at least 10 compounds, at least 12 compounds, at least 15compounds, or at least 20 compounds disclosed herein.

Also disclosed are oligosaccharides having the formula:

-   -   and salts thereof, wherein n is 1 or 0;    -   R^(a) is N₃, or NR^(4a)R^(4b),    -   wherein R^(4a) and R^(4b) are independently selected from H or        Z—X⁴,    -   wherein Z is null, C═O, or SO₂, and X⁴ is C₁₋₄alkyl,        O—C₁₋₄alkyl, C₁₋₄haloalkyl, O—C₁₋₄haloalkyl, C₁₋₄alkylaryl,        O—C₁₋₄alkylaryl, aryl, or O-aryl; or    -   R^(4a) and R^(4b) can together form a ring; or    -   R^(a) is a radical having the formula:

-   -   wherein one or both of R^(4c) or R^(4d) constitute a conjugated        payload;    -   R^(b) is N₃, or NR^(4a)R^(4b),    -   wherein R^(4a) and R^(4b) are independently selected from H or        Z—X⁴,    -   wherein Z is null, C═O, or SO₂, and X⁴ is C₁₋₄alkyl,        O—C₁₋₄alkyl, C₁₋₄haloalkyl, O—C₁₋₄haloalkyl, C₁₋₄alkylaryl,        O—C₁₋₄alkylaryl, aryl, or O-aryl; or    -   R^(4a) and R^(4b) can together form a ring; or R^(b) is a        radical having the formula:

-   -   wherein one or both of R^(4c) or R^(4d) constitute a conjugated        payload;    -   provided that R^(a) and R^(b) are not both NHC(O)CH₃;    -   R^(1a) is hydrogen or α-(L)-fucose, and    -   R³, R², and R^(g1) are independently selected from hydrogen or        further carbohydrate, for instance a monosaccharide or        oligosaccharide. Exemplary R³, R², and R^(g1) groups include        N-acetylglucosamine (GlcNAc), galactose (Gal), sialic acid        (Neu5Ac), and oligosaccharide comprising the same. Exemplary        sequences are depicted in Figure

In some instances R³ can be a moiety having the formula:

-   -   wherein R⁴ is selected from hydrogen or further carbohydrate,        for instance a monosaccharide or oligosaccharide. In some        instances R⁴ can be a moiety having the formula:

-   -   wherein R⁵ is selected from hydrogen or further carbohydrate,        for instance a monosaccharide or oligosaccharide. In some        instances R⁵ can be a moiety having the formula:

-   -   wherein R⁶ is selected from hydrogen or further carbohydrate,        for instance a monosaccharide or oligosaccharide. In some        instances R⁶ can be a moiety having the formula:

-   -   wherein R⁷ is selected from hydrogen or further carbohydrate,        for instance a monosaccharide or oligosaccharide. In some        instances R⁷ can be a moiety having the formula:

-   -   wherein R^(c3) is selected from hydrogen or conjugated payload,        and R^(c4) is selected from OH or conjugated payload.

In some instances R² can be a moiety having the formula:

-   -   wherein R⁴ is hydrogen or further carbohydrate, for instance a        monosaccharide or oligosaccharide. In some instances R⁴ can be a        moiety having the formula:

-   -   wherein R⁵ is hydrogen or further carbohydrate, for instance a        monosaccharide or oligosaccharide. In some instances R⁵ can be a        moiety having the formula:

-   -   wherein R⁶ is hydrogen or further carbohydrate, for instance a        monosaccharide or oligosaccharide. In some instances R⁶ can be a        moiety having the formula:

-   -   wherein R⁷ is hydrogen or further carbohydrate, for instance a        monosaccharide or oligosaccharide. In some instances R⁷can be a        moiety having the formula:

-   -   wherein R⁸ is hydrogen or a carbohydrate moiety having the        formula:

-   -   wherein R^(c3) is selected from hydrogen or conjugated payload,        and R^(c4) is selected from OH or conjugated payload.

R^(g1) is hydrogen or a carbohydrate moiety having the formula:

R² is hydrogen or a carbohydrate moiety having the formula:

-   -   wherein R⁴ is hydrogen or a carbohydrate moiety having the        formula:

-   -   wherein R⁵ is hydrogen or a carbohydrate moiety having the        formula:

-   -   wherein R⁶ is hydrogen or a carbohydrate moiety having the        formula:

-   -   wherein R⁷ is hydrogen or a carbohydrate moiety having the        formula:

-   -   wherein R⁸ is hydrogen or a carbohydrate moiety having the        formula:

-   -   wherein R^(c3) is selected from hydrogen or conjugated payload,        and R^(c4) is selected from OH or conjugated payload.

EXAMPLES

The following examples are for the purpose of illustration of theinvention only and are not intended to limit the scope of the presentinvention in any manner whatsoever.

¹H spectra were recorded on a 600 MHz Varian Inova or an Agilent 900 MHzDD2 spectrometer with a triple resonance (HCN) cryogenically cooledprobe spectrometer. Chemical shifts are reported in parts per million(ppm) relative to H1 and C1 of reducing N-acetylglucosamine which wereset to δ 5.08 and 78.02 repectivelly as the internal standard. NMR datais represented as follows: Chemical shift, multiplicity (s=singlet,d=doublet, t=triplet, dd=doublet of doublets, m=multiplet and/ormultiple resonances, br.=broad signal), J coupling, integration, andpeak identity. NMR signals were assigned based on ¹H NMR, gCOSY, gHSQC,zTOCSY, and NOESY experiments. Enzymatic reactions were monitored bymass spectrometry recorded on an Applied Biosystems SCIEX MALDI TOF/TOF5800 using 2,5-dihydroxybenzoic acid (DHB) as a matrix or a Shimadzu20AD UFLC LCMS-IT-TOF. Reagents were purchased from Sigma-Aldrich(unless otherwise noted) and used without further purification.HILIC-HPLC purification of compounds was performed on a Shimadzu 20ADUFLC LCMS-IT-TOF with a Waters XBridge BEH, Amide column, 5 μm, 10×250mm. HPLC grade acetonitrile and water were purchased from Fischer.Uridine 5′-diphosphogalactose diphosphate galactose (UDP-Gal) andcytidine-5′monophospho-N-acetylneuraminc acid (CMP-Neu5Ac) were bothpurchased from Roche, uridine 5′-diphospho-N-acetylglucosamine(UDP-GlcNAc) was purchased from Sigma-Aldrich, and guanosine5′-diphospho-β-L-fucose (GDP-Fuc) was purchased from Carbosynth.

2b. Extraction, Isolation and Trimming of SGP

Sialyl Glycopeptide (SGP, 5) Extraction

SGP (5) was extracted according to our previously reported procedure¹.In short, commercially available egg yolk powder (Natural Foods, Inc.,2.27 Kg) was suspended twice in 95% ethanol (4 L) and mechanicallystirred for 2 h at room temperature to remove lipids and other organicsoluble components. The filtrate was discarded and the insoluble powderwas suspended twice in aqueous ethanol (40% v/v ethanol, 3 L) solution.The insoluble material was discarded and the filtrate was concentratedunder reduced pressure at 40° C. The resulting translucent liquid waspurified using an active carbon/celite column (500 g of active carbonand 500 g celite). Impurities were removed by flushing the column with 3L of water (0.1% v/v TFA), 3 L of 5% acetonitrile in water (0.1% v/vTFA), and 3 L 10% acetonitrile in water (0.1% v/v TFA). The desiredglycopeptide was released from the column using a solution of 25%acetonitrile in water (0.1% v/v TFA), and fractions containing theproduct were pooled and dried under reduced pressure. The resultingwhite powder was subjected to size-exclusion chromatography (Bio-Rad®P-2, fine particle size 45-90 μm, column dimensions 5.0 cm×80 cm, 250 mLfractions) eluting with 0.1 M ammonium bicarbonate to yield SGP (5) as afluffy, white powder (1.82 g, or 0.8 mg SGP/g egg yolk powder).

Trimming and Modification of SGP to Prepare Glycosyl Asparagine-CBz 1¹Isolated SGP 5 (319 mg) was dissolved in 5 mL of Tris buffer (100 mM, pH8.0) containing 5 mM CaCl₂. Pronase from Streptomyces griseus(Sigma-Aldrich #P5147-1G, 150 mg) was added, and the reaction wasincubated for 5 days at 37° C. with shaking. The reaction was monitoredby ESI-MS and once complete the mixture was heated at 80° C. for 20 minfollowed by Pronase removal using an Amicon Ultra-10 (MWCO-10 k)centrifugal filter. The filtrate was lyophilized and purified bysize-exclusion chromatography (Bio-Rad P-2 BioGel, fine particle size45-90 μm, 2×80 cm), eluting with a 0.1 M ammonium bicarbonate solution.The fractions containing the glycosylated asparagine were pooled,lyophilized, and dissolved in 5 mL of water. To this mixture was addedK₂CO₃ (1.1 g), and CBzCl (0.54 g, 3.2 mmol) drop-wise. The heterogeneousmixture was stirred vigorously at room temperature until ESI-MSindicated complete installation of the CBz-protecting group (6). Thereaction was diluted with water (50 mL) and extracted with ethyl acetate(2×50 mL). The organic phase was discarded, and the aqueous phase waslyophilized and purified by size-exclusion chromatography using P-2BioGel eluting with a 0.1 M ammonium bicarbonate solution. The fractionscontaining 6 were pooled, lyophilized, and re-dissolved in 5 mL ofsodium acetate buffer (50 mM, pH 5.5) containing 5 mM CaCl₂. To thismixture was added neuraminidase from Clostridium perfringens (NewEngland Biolabs #P0720L, 40 μL, 2000 units) and the reaction wasincubated overnight at 37° C. with shaking at which time, ESI-MSindicated all the sialic acid residues had been removed. The pH of thereaction mixture was adjusted to 4.5 with acetic acid after which, BSA(5 mg) and β-galactosidase (200 μL, 800 units:) from Aspergillus niger(Megazyme #E-BGLAN) were added. The reaction was incubated at 37° C.with shaking overnight, after which another 150 μL of β-galactosidasewere added. The reaction was monitored by ESI-MS and once completegalactose removal was observed the enzymes were removed using an AmiconUltra-10 (MWCO-10 k) centrifugal filter. The filtrate was lyophilizedand purified by size-exclusion chromatography using P-2 BioGel elutingwith a 0.1 M ammonium bicarbonate solution. The fractions containing thetrimmed glycosyl asparagine-CBz were pooled, lyophilized, and dissolvedin 10 mL of MES buffer (100 mM, pH 7.3). To this mixture BSA (1 mg),calf intestine alkaline phosphatase (CIAP, 100 μL, 2 kU/mL), GDP-Fucose(75 mg), and FUT8 (200 μL, 1 mg/mL) were added and the reaction wasincubated overnight at 37° C. with shaking. The reaction was lyophilizedand purified by size-exclusion chromatography using P-2 BioGel elutingwith a 0.1 M ammonium bicarbonate solution. The fractions containing 1were pooled, lyophilized, and subjected to HILIC-HPLC (see section 2f)for final purification to give the compound 1 (74 mg, 39%).

2c. Expression and Purification of Enzymes

Recombinant Expression and Purification of PmGlmU

The gene sequence of Pasteurella multocidaN-acetylglucosamine-1-phosphate uridylyltransferase (PmGlmU) fromPasteurella multocida strain P-1059 (ATCC 15742) with a C-terminalHis₆-tag² were synthesized, ligated into a pET15b plasmid using NdeI andRhoI restriction sites, and transformed into E. coli BL21 (DE3) cells byGenscript. E. coli BL21 cells harboring the pET15b-PmGlmU plasmid werecultured in LB medium containing ampicillin (100 μg/mL) at 37° C. untilan OD_(600nm) of 0.8-1.0 was reached. Protein expression was induced bythe addition of isopropyl-1-thio-β-D-galactopyranoside (IPTG, finalconcentration 100 μM) and cultures where incubated at 20° C. withrigorous shaking for 18 h. The cells were harvested by centrifugation(4,000×g) at 4° C. for 20 min and the resulting pellet was resuspendedin lysis buffer (100 mM Tris-HCl, pH=8, containing 0.1% Triton X-100,lysozyme (100 μg/mL) and DNAse (5 μg/mL)). The cells were lysed bypassing the suspension twice through a French Press at 10,000 PSI and 4°C. and the lysate was clarified by centrifugation (10,000×g) at 4° C.for 45 min. Purification was performed by loading the supernatant onto aNi-NTA superflow column pre-equilibrated with binding buffer (10 mMimidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH=7.5). The column was washedwith washing buffer (40 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl,pH=7.5) and the PmGlmU enzyme was eluted with elution buffer (200 mMimidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH=7.5). Fractions containingpurified PmGlmU enzyme were combined and 10% glycerol was added forstorage at 4° C. From 1 L of culture medium 120-150 mg of PmGlmU wasobtained.

Human Glycosyl Transferase Expression and Purification

The catalytic domains of human glycosyl transferases (as shown in thetable below) were expressed as soluble, secreted fusion proteins bytransient transfection of HEK293 suspension cultures^(3,4). The codingregions were amplified from Mammalian Gene Collection clones, humantissue cDNAs, or generated by gene synthesis by a process that appendeda tobacco etch virus (TEV) protease cleavage site⁵ to the NH₂-terminalend of the coding region and attL1 and attL2 Gateway adaptor sites wereextended on the 5′ and 3′ terminal ends of the coding region duringtransfer to pDONR221 vector backbone⁴. The pDONR221 clones were thenrecombined via LR clonase reaction into a custom Gateway adapted versionof the pGEn2 mammalian expression vector⁴ to assemble a recombinantcoding region comprised of a 25 amino acid NH₂-terminal signal sequencefrom the T. cruzi lysosomal α-mannosidase⁶ followed by an 8×His tag, 17amino acid AviTag,⁷ “superfolder” GFP⁸, the nine amino acid sequenceencoded by attB1 recombination site, followed by the TEV proteasecleavage site and the respective glycosyltransferase catalytic domaincoding region.

Suspension culture HEK293 cells (Freestyle 293-F cells, LifeTechnologies, Grand Island, N.Y.) were transfected as previouslydescribed^(3,4) and the culture supernatant was subjected to Ni²⁺-NTAsuperflow chromatography (Qiagen, Valencia, Calif.). Enzyme preparationswere eluted with 300 mM imidazole, concentrated by ultrafiltration, andsubjected to gel filtration on a Superdex 75 column (GE Healthcare)preconditioned with a buffer containing 20 mM HEPES, pH 7.0, 100 mMNaCl, 10% glycerol, 0.05% Na azide. Peak fractions were pooled andconcentrated to ˜1 mg/mL using an ultrafiltration pressure cell membrane(Millipore, Billerica, Mass.) with a 10 kDa molecular weight cutoff.

2d. UDP-GlcNTFA Preparation

Procedure for the One-Pot Three-Enzyme Preparation of UDP-GlcNTFA (4)

GlcNTFA⁹ (162 mg, 589 μmol), ATP (390 mg, 707 μmol) and UTP (390 mg, 707μmol) were dissolved in 59 mL of 100 mM Tris-HCl buffer (pH=8.0)containing 10 mM MgCl₂. To this solution was added Bifidobacteriumlongum N-acetylhexosamine 1-kinase (NahK, 14 μg/μmol substrate),Pasteurella multocida N-acetylglucosamine-1-phosphateuridylyltransferase (PmGlmU, 17 μg/μmol substrate) and Pasteurellamultocida inorganic pyrophosphatase (PmPpA, 7 μg/μmol substrate), andthe reaction mixture was incubated overnight at 37° C. with gentleshaking. Reaction progress was monitored by ESI-TOF MS, and oncecomplete 59 mL of cold ethanol was added and the mixture was incubatedat 4° C. for 1 h. The reaction mixture was centrifuged and thesupernatant was removed, concentrated, and purified by a P2 BioGelcolumn using 0.1 M NH₄HCO₃ as eluent, followed by silica gel columnchromatography (4:2:1 EtOAc/MeOH/H₂O) afforded UDP-GlcNTFA 4 (273 mg,70%) as a white solid². ¹H NMR (500 MHz, D₂O): δ7.97 (d, J=8.1 Hz, 1 H,H6-Uridine), 5.99 (d, J=4.5 Hz, 1 H, H1-Ribose), 5.98 (d, J=8.0 Hz, 1 H,H5-Uridine), 5.63 (dd, J=7.0, 3.3 Hz, 1 H, H1-GlcNTFA), 4.42-4.34 (m, 2H, H2-Ribose, H3-Ribose), 4.33-4.28 (m, 1 H, H4-Ribose), 4.24 (dd,J=4.5, 2.7 Hz, 1 H, H5-Ribose), 4.21 (dd, J=5.6, 3.0 Hz, 1 H,H5′-Ribose), 4.12 (dt, J=10.8, 2.9 Hz, 1 H, H2-GlcNTFA), 4.00-3.95 (m, 2H, H3-GlcNTFA, H4-GlcNTFA), 3.89 (dd, J=12.5, 2.3 Hz, 1 H, H6-GlcNTFA),3.83 (dd, J=12.6, 4.3 Hz, 1 H, H6′-GlcNTFA), 3.62-3.59 (m, 1 H,H5-GlcNTFA. ¹³C NMR (76 MHz, D₂O): δ141.6 (C6-Uridine), 102.5(C5-Uridine), 93.7 (C1-GlcNTFA), 88.5 (C1-Ribose), 83.0 (C4-Ribose),73.7 (C2-Ribose), 73.0 (C3-GlcNTFA), 70.1 (C4-GlcNTFA), 69.5(C3-Ribose), 69.4 (C5-GlcNTFA), 64.9 (C5-Ribose), 60.1 (C6-GlcNTFA),54.3 (C2-GlcNTFA). ESI-MS m/z calcd for C₁₇H₂₃F₃N₃O₁₇P₂, [M-H]⁻:660.0460, found 660.0417.

2e. General Protocols for Enzymatic Reactions and Glycosyl AsparagineModification General Procedure for the Installation of core α1,6 Fucusing FUT8. Glycosyl asparagine acceptor A2-Asn-Cbz (79 mg, 55 μmol) andGDP-Fuc (75 mg, 111.8 μmol) were dissolved at a final acceptorconcentration of 10 mM in a MES buffered solution (100 mM, pH 7.5)containing BSA (1% total volume, stock solution=10 mg mL⁻¹). Calfintestine alkaline phosphatase (CIAP, 1% total volume, stock solution=1kU mL⁻¹) and FUT8 (40 μg/μmol acceptor) were added, and the reactionmixture was incubated overnight at 37° C. with gentle shaking. Reactionprogress was monitored by ESI-TOF MS and if starting material remainedafter 18 h another portion of FUT8 was added until no starting materialcould be detected. The reaction mixture was centrifuged over a Nanosep®Omega ultrafiltration device (10 kDa MWCO) to remove reaction proteinsand the filtrate was lyophilized. Purification by HPLC using a HILICcolumn (supporting information 2f) provided desired product as a whitefluffy solid (74 mg, 85%).

General Procedure for the Installation of β1,3 GlcNAc using B3GNT2

Glycosyl asparagine acceptor (1 eq) and UDP-GlcNAc (1.5 eq) weredissolved to provide a final acceptor concentration of 2-5 mM in a HEPESbuffered solution (50 mM, pH 7.3) containing KCl (25 mM), MgCl₂ (2 mM)and DTT (1 mM). Calf intestine alkaline phosphotase (CIAP, 1% totalvolume, 1 kU mL⁻¹) and B3GNT2 (1% wt/wt relative to acceptor substrate)were added, and the reaction mixture was incubated overnight at 37° C.with gentle shaking. Reaction progress was monitored by MALDI-TOF MS orESI-TOF MS, and if starting material remained after 18 h another portionof B3GNT2 was added until no starting material could be detected. Thereaction mixture was centrifuged over a Nanosep® Omega ultrafiltrationdevice (10 kDa MWCO) to remove reaction proteins and the filtrate waslyophilized. Purification by HILIC HPLC (see section 2f) or P2size-exclusion column chromatography provided the desired product.

General procedure for the installation of β1,2-GlcNTFA using MGAT1.Glycosyl asparagine acceptor Man3-Asn-Cbz (5.0 mg, 4.3 μmol) andUDP-GlcNTFA (5.7 mg, 8.6 μmol) were dissolved at a final acceptorconcentration of 10 mM in a MES buffered solution (100 mM, pH 6.5)containing MnCl₂ (10 mM) and BSA (1% total volume, stock solution=10 mgmL⁻¹). Calf intestine alkaline phosphatase (CIAP, 1% total volume) andMGAT1 (40 μg/μmol acceptor) were added, and the reaction mixture wasincubated overnight at 37° C. with gentle shaking. Reaction progress wasmonitored by ESI-TOF MS and if starting material remained after 18 hanother portion of MGAT1 was added until no starting material could bedetected. The reaction mixture was centrifuged over a Nanosep® Omegaultrafiltration device (10 kDa MWCO) to remove reaction proteins and thefiltrate was lyophilized. Purification by HPLC using a HILIC columnprovided desired product as a white fluffy solid (4.9 mg, 81%).

General procedure for the installation of β1,2-GlcNTFA using MGAT2.Glycosyl asparagine acceptor Man3A1-Asn-Cbz (3.0 mg, 2.2 μmol) andUDP-GlcNTFA (3 mg, 4.5 μmol) were dissolved at a final acceptorconcentration of 5 mM in a MES buffered solution (100 mM, pH 7.5)containing BSA (1% total volume). CIAP (1% total volume) and MGAT2 (400μg/μmol acceptor) were added, and the reaction mixture was incubatedovernight at 37° C. with gentle shaking. Reaction progress was monitoredby ESI-TOF MS, and if starting material remained after 18 h anotherportion of MGAT2 was added until no starting material could be detected.The reaction mixture was centrifuged over a Nanosep® Omegaultrafiltration device (10 kDa MWCO) to remove reaction proteins, andthe filtrate was lyophilized. Purification by HPLC using a HILIC columnprovided the desired product Man3A2-Asn-Cbz as a white fluffy solid (2.7mg, 76%).

General procedure for the installation of β1,6-GlcNTFA using MGAT5.Glycosyl asparagine acceptor 1 (17.6 mg, 10.2 μmol) and UDP-GlcNTFA(13.5 mg, 20.4 μmol) were dissolved at a final acceptor concentration of10 mM in a sodium cacodylate buffered solution (100 mM, pH 6.5)containing MnCl₂ (10 mM) and BSA (1% total volume, stock solution=10 mgmL⁻¹). Calf intestine alkaline phosphatase (CIAP, 1% total volume, stocksolution=1 kU mL⁻¹) and MGAT5 (40 μg/μmol acceptor) were added, and thereaction mixture was incubated overnight at 37° C. with gentle shaking.Reaction progress was monitored by MALDI-TOF MS and if starting materialremained after 18 h another portion of MGAT5 was added until no startingmaterial could be detected. The reaction mixture was centrifuged over aNanosep® Omega ultrafiltration device (10 kDa MWCO) to remove reactionproteins and the filtrate was lyophilized. Purification by HPLC using aHILIC column (supporting information 2f) provided desired product 14 asa white fluffy solid (18.6 mg, 92%).

General procedure for the installation of β1,4-GlcNTFA using MGAT4B.Glycosyl asparagine acceptor 2 (4.0 mg, 2.1 μmol) and UDP-GlcNTFA (2.75mg, 4.2 μmol) were dissolved at a final acceptor concentration of 5 mMin a Tris buffered solution (100 mM, pH 7.5) containing MnCl₂ (5 mM) andBSA (1% total volume). CIAP (1% total volume) and MGAT4B (400 μg/μmolacceptor) were added, and the reaction mixture was incubated overnightat 37° C. with gentle shaking. Reaction progress was monitored byESI-TOF MS, and if starting material remained after 18 h another portionof MGAT4B was added until no starting material could be detected. Thereaction mixture was centrifuged over a Nanosep® Omega ultrafiltrationdevice (10 kDa MWCO) to remove reaction proteins, and the filtrate waslyophilized. Purification by HPLC using a HILIC column (supportinginformation 2f) provided the desired product S6 as a white fluffy solid(3.8 mg, 85%).

General Procedure for the Installation of β1,4 Gal using B4GALT1

Glycosyl asparagine acceptor (1 eq) and UDP-Gal (1.5 eq per Gal to beadded) were dissolved to a provide an acceptor concentration of 2-5 mMin a Tris buffered solution (100 mM, pH 7.5) containing MnCl₂ (10 mM)and BSA (1% total volume). CIAP (1% volume total) and B4GALT1 (1% wt/wtrelative to acceptor substrate) were added, and the reaction mixture wasincubated overnight at 37° C. with gentle shaking. Reaction progress wasmonitored by MALDI-TOF MS or ESI-TOF MS, and if starting materialremained after 18 h another portion of B4GALT1 was added until nostarting material could be detected. The reaction mixture wascentrifuged over a Nanosep® Omega ultrafiltration device (10 kDa MWCO)to remove reaction proteins and the filtrate was lyophilized.Purification by HILIC HPLC (see section 2f) or P2 size-exclusion columnchromatography provided the desired product.

General Procedure for the Installation of α1,3 Fuc using FUT5

Glycosyl asparagine acceptor (1 eq) and GDP-Fuc (1.5 eq per Fuc to beadded) were dissolved at a final acceptor concentration of 2-5 mM in aTris buffered solution (50 mM, pH 7.3) containing MnCl₂ (10 mM). CIAP(1% total volume) and FUT5 (1% wt/wt) were added, and the reactionmixture was incubated overnight at 37° C. with gentle shaking. Reactionprogress was monitored by MALDI-TOF MS or ESI-TOF MS, and if startingmaterial remained after 18 h another portion of FUT5 was added until nostarting material could be detected. The reaction mixture wascentrifuged over a Nanosep® Omega ultrafiltration device (10 kDa MWCO)to remove reaction proteins and the filtrate was lyophilized.Purification by HILIC HPLC (see section 2f) or P2 size-exclusion columnchromatography provided the desired product.

General Procedure for the Installation of α2,3 Neu5Ac using ST3GAL4

Glycosyl asparagine acceptor (1 eq) and CMP-Neu5Ac (1.5 eq) weredissolved at a final acceptor concentration of 2-5 mM in a sodiumcacodylate buffered solution (50 mM, pH 7.2) containing BSA (1% totalvolume). CIAP (1% volume total) and ST3GAL4 (1% wt/wt relative toacceptor substrate) were added, and the reaction mixture was incubatedovernight at 37° C. with gentle shaking. Reaction progress was monitoredby ESI-TOF MS, and if starting material remained after 18 h anotherportion of ST3GAL4 was added until no starting material could bedetected. The reaction mixture was centrifuged over a Nanosep® Omegaultrafiltration device (10 kDa MWCO) to remove reaction proteins, andthe filtrate was lyophilized. Purification by HILIC HPLC (see section2f) or P2 size-exclusion column chromatography provided the desiredproduct.

General Procedure for the Selective Installation of Terminal α2,6 Neu5Acusing ST6GAL1

Glycosyl asparagine (1 eq) and CMP-Neu5Ac (1.1 eq) were dissolved at afinal acceptor concentration of 2-5 mM in a sodium cacodylate bufferedsolution (100 mM, pH 6.5) containing BSA (1% volume total). CIAP (1%volume total) and ST6GAL1 (1% wt/wt relative to acceptor substrate) wereadded, and the reaction mixture was incubated overnight at 37° C. withgentle shaking. The reaction mixture was centrifuged over a Nanosep®Omega ultrafiltration device (10 kDa MWCO) to remove reaction proteins,and the filtrate was lyophilized. Purification by HILIC HPLC (seesection 2f) or P2 size-exclusion column chromatography provided thedesired product.

General Procedure for the Selective Cleavage of Galactose using E. coliβ-galactosidase¹⁰

Glycosyl asparagine was dissolved at a concentration of 5 mM in a Trisbuffered solution (50 mM, pH 7.3) containing 5 mM MgCl₂. To thissolution was added 50 U/μmol glycosyl asparagine of E. coliβ-galactosidase (Sigma-Aldrich #, G5635) and the mixture was incubatedovernight at 37° C. The reaction mixture was centrifuged using aNanosep® Omega ultrafiltration device (10 kDa MWCO) to remove the enzymeand the filtrate was lyophilized Purification by HILIC HPLC (see section2f) or P2 size-exclusion column chromatography provided the desiredproduct.

General procedure for removal of TFA protecting group of an N-glycan.The GlcNTFA moiety of S6 was converted to GlcNH₂ by dissolving thesubstrate (3.8 mg, 1.8 μM) in H₂O to a final concentration of 10 mM. ThepH of the solution was adjusted to 10 using μL aliquots 1 M NaOH. Thereaction mixture was incubated overnight at 37° C. with gentle shaking.Progress of the reaction was monitored by MALDI-TOF MS and once completethe solvent was removed by lyophilization. The reaction was neutralizedby μL aliquots of 1 M acetic acid and purified by P2 size-exclusionchromatography eluting with 50 mM ammonium bicarbonate to yield thedesired target 2 as a white fluffy solid (3.5 mg, 92%).

General procedure for the conversion of GlcNH₂ to GlcN₃. Substrate 15(9.3 mg, 5 μmol, 1 eq) was dissolved in water (1.6 mL) and to thissolution was added imidazole-1-sulfonyl azide hydrogen sulfate (13.4 mg,50 mol), K₂CO₃ (6.8 mg, 50 μmol) and catalytic CuSO₄.5H₂O. The reactionmixture was incubated overnight at 37° C. with gentle shaking. Reactionprogress was monitored by MALDI-TOF MS and if starting materialremained, an additional ½ portion of the imidazole-1-sulfonyl azidehydrogen sulfate, K₂CO₃, and CuSO₄ was added until no starting materialcould be observed. The reaction solvent was removed by lyophilizationand the salts were removed by P2 size-exclusion chromatography elutingwith 50 mM ammonium bicarbonate to yield 23 as a white fluffy solid (7.2mg, 76%).

General procedure for reduction of GlcN₃. Intermediate 27 (2.3 mg, 0.66μmol, 1 eq) was dissolved in a solution of 9:1 pyridine/triethylamine togive a final concentration of 5 mM. The mixture was vortexed until allsolids dissolved and 10 eq. 1,3-dithiolpropane (0.7 mg, 6.6 μmol, 10 eq)were added in one portion. The reaction mixture was kept at 37° C. wasuntil no azide could be detected by ESI-TOF-MS. Reaction was carriedforward to acetylate the amine without further purification.

General procedure for amine acetylation. 18 (1.3 mg, 0.5 μmol, 1 eq) wasdissolved in water to a final concentration of 2 mM. The pH was adjustedto 8 using μL aliquots of 1M NaOH. To this solution was added solidAcOSu (0.7 mg, 5 μmol, 10 eq) in one portion. The reaction mixture wasvortexed vigorously until all solids were dissolved. The reaction waskept at 37° C. until full acetylation was observed by ESI-TOF-MS. In theevent starting amine was detected, additional AcOSu (5 eq) was addeduntil complete conversion was observed. The reaction was lyophilized andpurified by HPLC using a HILIC column (supporting information 2f) toafford 19 as a white fluffy solid (0.9 mg, 67%).

2f. General Protocols for HILIC-HPLC Purification

HILIC-HPLC Purification Conditions for Glycosyl Asparagine Targets

Semi-preparative HILIC-HPLC was performed on a Shimadzu LC-ESI-IT-TOFwith a Waters XBridge BEH, Amide column, 5 μm, 10×250 mm at a flow rateof 2.3 mL/min, injection volume of 100 μL (10-20 mg/mL), with 1% of theflow is diverted to the ESI-MS detector using a splitter. Mobile phase Awas 10 mM ammonium formate in water, adjusted to pH 4.5 with formicacid; mobile phase B was 90% aceteonitrile with 10% 10 mM ammoniumformate in water (pH=4.5). The general condition using a linear gradientis as follows:

Time (min) A (%) B (%) 0 20 80 40 55 45 45 80 20 55 20 80 60 20 80

1 was purified using a linear gradient with the following conditions:

Time (min) A (%) B (%) 0 20 80 60 40 60 70 50 50 71 80 20 80 80 20 85 2080 90 20 80

The compositions and methods of the appended claims are not limited inscope by the specific compositions and methods described herein, whichare intended as illustrations of a few aspects of the claims and anycompositions and methods that are functionally equivalent are intendedto fall within the scope of the claims. Various modifications of thecompositions and methods in addition to those shown and described hereinare intended to fall within the scope of the appended claims. Further,while only certain representative compositions and method stepsdisclosed herein are specifically described, other combinations of thecompositions and method steps also are intended to fall within the scopeof the appended claims, even if not specifically recited. Thus, acombination of steps, elements, components, or constituents may beexplicitly mentioned herein or less, however, other combinations ofsteps, elements, components, and constituents are included, even thoughnot explicitly stated. The term “comprising” and variations thereof asused herein is used synonymously with the term “including” andvariations thereof and are open, non-limiting terms. Although the terms“comprising” and “including” have been used herein to describe variousembodiments, the terms “consisting essentially of” and “consisting of”can be used in place of “comprising” and “including” to provide for morespecific embodiments of the invention and are also disclosed. Other thanin the examples, or where otherwise noted, all numbers expressingquantities of ingredients, reaction conditions, and so forth used in thespecification and claims are to be understood at the very least, and notas an attempt to limit the application of the doctrine of equivalents tothe scope of the claims, to be construed in light of the number ofsignificant digits and ordinary rounding approaches.

1. A compound having the formula:

or a salt thereof, wherein R^(MG2), R^(MG3), R^(MG4), and R^(MG5) areeach independently chosen from H, GlcNAc, or modified GlcNAc R¹ is OH,or a residue having the formula:

R^(oz) is H, C₁₋₄alkyl, aryl, CF₃, or CCl₃, n is 1 or 0; R^(fa) is H ora fucose residue having the structure:

R² is OR^(c), NR^(2n)OR^(c); or NR^(2n)R^(c) and R³ is OR^(c) or R^(c);wherein: R^(2n) is H or C₁₋₄alkyl; R^(c) is X^(p)H, X^(p)C₁₋₈alkyl,X^(p)C₁₋₈alkylaryl, X^(p)aryl, X^(p)fluorescent marker, or an amino acidresidue having the formula:

wherein R^(aa1) and R^(aa2) are selected from H, protecting group andadditional amino acid residues; R³ is X^(p)C₁₋₈alkyl,X^(p)C₁₋₈alkylaryl, or X^(p)aryl; wherein X^(p) is null or—(CH₂CH₂O)_(z)—, wherein z is 1-500. R⁴ is N₃, or NR^(4a)R^(4b),wherein: R^(4a) and R^(4b) are the same or different, and are H or Z—X⁴wherein Z is null, C═O, or SO₂, and X⁴ is C₁₋₄alkyl, O—C₁₋₄alkyl,C₁₋₄haloalkyl, O—C₁₋₄haloalkyl, C₁₋₄alkylaryl, O—C₁₋₄alkylaryl, aryl, orO-aryl; or R^(4a) and R^(4b) can together form a ring; or R⁴ is aradical having the formula:

wherein one or both of R^(4c) or R^(4d) constitute a conjugated payload.2. The compound according to claim 1, wherein R^(aa1) and R^(aa2) areindependently selected from H and Z^(a)—X^(a) wherein Z^(a) is null,C═O, SO₂, and X^(a) can be C₁₋₄alkyl, O—C₁₋₄alkyl, C₁₋₄haloalkyl,O—C₁₋₄haloalkyl, C₁₋₄alkylaryl, O—C₁₋₄alkylaryl, aryl, or O-aryl.
 3. Thecompound according to claim 1, wherein R^(aa1) is Cbz (carboxybenzylether), and R^(aa2) is H.
 4. (canceled)
 5. The compound according toclaim 1, wherein R¹ has formula:


6. (canceled)
 7. The compound according to claim 1, wherein R¹ is aresidue having the formula:


8. (canceled)
 9. The compound according to claim 1, wherein R^(c) hasthe formula —(CH₂)_(nc)R^(im), wherein nc is an integer from 1-8, andR^(im) —NH₂, —SH, —OH, —COOH, —N₃, C≡CH, a Michael acceptor, SO₃, OSO₃,or a biotin residue having the formula:

wherein X^(bt) is selected from null, O, NH, or S.
 10. (canceled) 11.The compound according to claim 1, wherein R^(c) is selected fromcoumarins, rhodamines, fluoresceins, cyanines, eosins, and erythrosins.12. The compound according to claim 1 any preceding claim, wherein R^(c)is a C₁₋₈alkylaryl or aryl.
 13. The compound according to claim 1,wherein R^(c) is a group having the formula:

wherein np is from 0-50, ns is from 2-7, and ns′ is from 2-5. 14.(canceled)
 15. (canceled)
 16. The compound according to claim 1, whereinR^(MG2), R^(MG3), R^(MG4), and R^(MG5) are each hydrogen.
 17. Thecompound according to claim 1, having the formula:

R⁵ is N₃ or NR^(5a)R^(5b), wherein R^(5a) and R^(5b) are the same ordifferent, and are H or Z—X⁵ wherein Z is null, C═O, or SO₂, and X⁴ isC₁₋₄alkyl, O—C₁₋₄alkyl, C₁₋₄haloalkyl, O—C₁₋₄haloalkyl, C₁₋₄alkylaryl,O—C₁₋₄alkylaryl, aryl, or O-aryl; or R^(5a) and R^(5b) can together forma ring; or R⁵ is a radical having the formula:

wherein one or both of R^(5c) or R^(5d) constitute a conjugated payload.18. The compound according to claim 1, wherein the R⁵ bearing residue isan isotopically enriched GlcNAc or modified GlcNAc.
 19. The compoundaccording to claim 1, having the formula:

R⁶ is hydrogen or a residue having the formula:

wherein R⁹ is N₃ or NR^(9a)R^(9b), wherein R^(9a) and R^(9b) are thesame or different, and are H or Z—X⁹ wherein Z is null, C═O, SO₂, and X⁹can be C₁₋₄alkyl, O—C₁₋₄alkyl, C₁₋₄haloalkyl, O—C₁₋₄haloalkyl,C₁₋₄alkylaryl, O—C₁₋₄alkylaryl, aryl, or O-aryl; or R^(9a) and R^(9b)can together form a ring; or R⁹ is a radical having the formula:

wherein one or both of R^(9c) or R^(9d) constitute a conjugated payload;R⁷ is hydrogen or a residue having the formula:

wherein R¹⁰ is N₃ is NR^(10a)R^(10b), wherein R^(10a) and R^(10b) arethe same or different, and can be H, Z—X¹⁰ wherein Z is null, C═O, orSO₂, and X⁴ is C₁₋₄alkyl, O—C₁₋₄alkyl, C₁₋₄haloalkyl, O—C₁₋₄haloalkyl,C₁₋₄alkylaryl, O—C₁₋₄alkylaryl, aryl, or O-aryl; or R^(10a) and R^(10b)can together form a ring; or R¹⁰ can be a radical having the formula:

wherein one or both of R^(10c) or R^(10d) constitute a conjugatedpayload; R¹² is hydrogen or a residue having the formula:

wherein R¹³ is hydrogen or a residue having the formula:

wherein R^(13b) is selected from H and a conjugated cargo moiety, andR^(13a) is selected from OH and a conjugated cargo moiety; R¹⁴ ishydrogen or a residue having the formula:

wherein R^(14b) is selected from H and a conjugated cargo moiety, andR^(14a) is selected from OH and a conjugated cargo moiety; R⁸ ishydrogen or a residue having the formula:

wherein R¹¹ is N₃ or NR^(11a)R^(11b), wherein R^(11a) and R^(11b) arethe same or different, and are H or Z—X¹¹ wherein Z is null, C═O, orSO₂, and X¹¹ is C₁₋₄alkyl, O—C₁₋₄alkyl, C₁₋₄haloalkyl, O—C₁₋₄haloalkyl,C₁₋₄alkylaryl, O—C₁₋₄alkylaryl, aryl, or O-aryl; or R^(11a) and R^(11b)can together form a ring; or R¹¹ can be a radical having the formula:

wherein one or both of R^(11c) or R^(11d) constitute a conjugatedpayload.
 20. (canceled)
 21. A method for preparing an oligosaccharide,comprising the steps of glycosylating an oligosaccharide of Formula (1):

R¹ is OH or a residue having the formula:

R^(oz) is H, C₁₋₄alkyl, aryl, CF₃, or CCl₃, n is 1 or 0; R^(fa) is H ora fucose residue having the structure: is OR^(c) or NR^(2n)OR^(c);NR^(2n)R^(c); and R³ is OR^(c) or R^(c); wherein: R^(2n) is H orC₁₋₄alkyl; R^(c) is X^(p)H, X^(p)C₁₋₈alkyl, X^(p)C₁₋₈alkylaryl,X^(p)aryl, X^(p)fluorescent marker, or an amino acid residue having theformula:

wherein R^(aa1) and R^(aa2) are H, protecting group or additional aminoacid residues; R³ is X^(p)C₁₋₈alkyl, X^(p)C₁₋₈alkylaryl, or X^(p)aryl;wherein X^(p) ise null or —(CH₂CH₂O)_(z)—, wherein z is 1-500. with amodified GlcNAc donor of Formula (2):

wherein R^(a) is CX₃, CHX₂, CH₂X, CH₃, OBn, wherein X is in each caseindependently selected from F, Cl, Br, and I, in the presence of MGAT1,to give an oligosaccharide having the Formula (3):

wherein R⁴ is NHC(O)R^(a).
 22. The method according to claim 21, furthercomprising converting R⁴ to NH₂ or N₃.
 23. The method according to claim22, comprising the steps of glycosylating the oligosaccharide of Formula(3) with a modified GlcNAc donor of formula (2d):

wherein R^(a) is CX₃, CHX₂, CH₂X, CH₃, or OBn, wherein X is in each caseindependently selected from F, Cl, Br, and I, in the presence of MGAT,to give an oligosaccharide having the Formula (4):

wherein R⁵ is NHC(O)R^(a) and R⁴ and R⁵ are not the same.
 24. The methodaccording to claim 23, comprising converting R⁵ to NH₂ or N₃.
 25. Themethod according to claim 23, wherein the modified GlcNAc donor ofFormula (2d) is ¹³C enriched. 26-46. (canceled)
 47. A compound havingthe formula:

or a salt thereof, wherein n is 1 or 0; R^(a) is N₃, or NR^(4a)R^(4b),wherein R^(4a) and R^(4b) are independently selected from H or Z—X⁴,wherein Z is null, C═O, or SO₂, and X⁴ is C₁₋₄alkyl, O—C₁₋₄alkyl,C₁₋₄haloalkyl, O—C₁₋₄haloalkyl, C₁₋₄alkylaryl, O—C₁₋₄alkylaryl, aryl, orO-aryl; or R^(4a) and R^(4b) can together form a ring; or R^(a) is aradical having the formula:

wherein one or both of R^(4c) or R^(4d) constitute a conjugated payload;R^(b) is N₃, or NR^(4a)R^(4b), wherein R^(4a) and R^(4b) areindependently selected from H or Z—X⁴, wherein Z is null, C═O, or SO₂,and X⁴ is C₁₋₄alkyl, O—C₁₋₄alkyl, C₁₋₄haloalkyl, O—C₁₋₄haloalkyl,C₁₋₄alkylaryl, O—C₁₋₄alkylaryl, aryl, or O-aryl; or R^(4a) and R^(4b)can together form a ring; or R^(b) is a radical having the formula:

wherein one or both of R^(4c) or R^(4d) constitute a conjugated payload;provided that R^(a) and R^(b) are not both NHC(O)CH₃; R^(1a) is hydrogenor α-(L)-fucose, and R³, R², and R^(g1) are independently selected fromhydrogen or additional carbohydrate.